Anti-C2/C2a inhibitors of complement activation

ABSTRACT

The invention relates to C2a inhibitors, which bind to C2a and block the functional activity of C2a in complement activation. The inhibitors include antibody molecules, as well as homologues, analogues and modified or derived forms thereof, including immunoglobulin fragments like Fab, F(ab′) 2  and Fv, small molecules, including peptides, oligonucleotides, peptidomimetics and organic compounds. A monoclonal antibody, which bound to C2a and blocked its ability to activate complement was generated and designated 175-62. The hybridoma producing this antibody was deposited at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, under Accession Number PTA-1553.

This application claims priority to U.S. Provisional Application No.60/191,429, filed on Mar. 23, 2000.

FIELD OF THE INVENTION

The present invention relates to inhibitor molecules specific tocomplement C2 and its activation fragment C2a, the use of such inhibitormolecules to block complement activation via the classical pathway andthe lectin pathway, treatment of diseases associated with excessivecomplement activation, and the diagnostic determination of the amount ofC2a present in a biological sample.

BACKGROUND OF THE INVENTION

The complement system is part of the innate immune system and consistsof many components that act in a cascade fashion. This system plays acentral role in both the clearance of immune complexes and the immuneresponse to infectious agents, foreign antigens, virus-infected cellsand tumor cells. However, complement is also involved in pathologicalinflammation and in autoimmune diseases. Therefore, inhibition ofexcessive or uncontrolled activation of the complement cascade couldprovide clinical benefit to patients with such diseases and conditions.

The complement system can be activated in three ways, either by one ofthe two primary activation pathways, designated the classical and thealternative pathways (V. M. Holers, In Clinical Immunology: Principlesand Practice, ed. R. R. Rich, Mosby Press, 1996, 363-391), or by a thirdpathway, the lectin pathway activated by mannan-binding lectin (MBL) (M.Matsushita, Microbiol. Immunol., 1996, 40: 887-893; M. Matsushita etal., Immunobiol., 1998, 199: 340-347; T. Vorup-Jensen et al.,Immunobiol., 1998, 199: 348-357).

The classical pathway is a calcium/magnesium-dependent cascade, which isnormally activated by the formation of antigen-antibody complexes. C1,the first enzyme complex in the cascade, is a pentamolecular complexconsisting of C1q, 2 C1r molecules, and 2 C1s molecules. This complexbinds to an antigen-antibody complex through the C1q domain to initiatethe cascade. Once activated, C1s cleaves C4 resulting in C4b, which inturn binds C2. C2 is cleaved by C1s, resulting in the activated form,C2a, bound to C4b and forming the classical pathway C3 convertase.

The alternative pathway is a magnesium-dependent cascade and isantibody-independent. This pathway is activated by a variety of diversesubstances including, e.g., cell wall polysaccharides of yeast andbacteria, and certain biopolymer materials. When the C3 protein binds oncertain susceptible surfaces, it is cleaved to yield C3b thus initiatingan amplification loop.

The lectin pathway involves complement activation by MBL through twoserum serine proteases designated MASP-1 and MASP-2 (as opposed to C1rand C1s in the classical complement pathway). Like the classicalcomplement pathway, the lectin complement pathway also requires C4 andC2 for activation of C3 and other terminal components further downstreamin the cascade (C. Suankratay et al., J. Immunol., 1998, 160: 3006-3013;Y. Zhang et al., Immunopharmacol., 1999, 42: 81-90; Y. Zhang et al.,Immunol., 1999, 97:686-692; C. Suankratay et al., Clin. Exp. Immunol.,1999, 117: 442-448). Alternative pathway amplification is also requiredfor lectin pathway hemolysis in human serum (C. Suankratay et al., J.Immunol., 1998, 160: 3006-3013; C. Suankratay et al., Clin. Exp.Immunol., 1998, 113: 353-359). In short, Ca⁺⁺-dependent binding of MBLto a mannan-coated surface triggers activation of C3 following C4 and C2activation, and the downstream activation of C3 and the terminalcomplement components then require the alternative complement pathwayfor amplification.

Activation of the complement pathway generates biologically activefragments of complement proteins, e.g. C3a, C4a and C5a anaphylatoxinsand sC5b-9 membrane attack complex (MAC), which mediate inflammatoryactivities involving leukocyte chemotaxis, activation of macrophages,neutrophils, platelets, mast cells and endothelial cells, vascularpermeability, cytolysis, and tissue injury (R. Schindler et al., Blood,1990, 76: 1631-1638; T. Wiedmer, Blood, 1991, 78: 2880-2886; M. P.Fletcher et al., Am. J. Physiol., 1993, 265: H1750-1761).

C2 is a single-chain plasma protein of molecular weight of 102 kD, whichis specific for the classical and the lectin complement pathways.Membrane bound C4b expresses a binding site which, in the presence ofMg⁺⁺, binds the proenzyme C2 near its amino terminus and presents it forcleavage by C1s (for the classical complement pathway) or MASP-2 (forthe lectin complement pathway) to yield a 30 kD amino-terminal fragment,C2b, and a 70 kD carboxy-terminal fragment, C2a (S. Nagasawa et al.,Proc. Natl. Acad. Sci. (USA), 1977, 74: 2998-3003). The C2b fragment maybe released or remain loosely attached to C4b. The C2a fragment remainsattached to C4b to form the C4b2a complex, the catalytic components ofthe C3 and C5 convertases of the classical and the lectin complementpathways. The enzymatic activity in this complex resides entirely inC2a, C4b acting to tether C2a to the activating surface.

Monoclonal antibodies (MAbs) to human C2 and its fragments C2a and C2bwere made by immunizing mice with purified human C2 (E. I. Stenbaek etal., Mol. Immunol., 1986, 23: 879-886; T. J. Oglesby et al., J.Immunol., 1988, 141: 926-932). The novel anti-C2a MAbs of the presentinvention were made by immunizing mice with purified human C2a fragmentand were shown to have inhibitory activity against the classical pathwaycomplement activation (see below). These anti-C2a MAbs are distinct fromthe known anti-C2b MAbs (see T. J. Oglesby et al., J. Immunol., 1988,141: 926-932) because bind latter to different segments of C2 andinhibit the classical complement pathway by interfering the interactionbetween C2 and C4 (T. J. Oglesby et al., J. Immunol., 1988, 141:926-932). By virtue of this inhibition, the anti-C2a MAbs of the presentinvention are the first Mab demonstrated to be effective in inhibitingthe classical complement pathway.

Targeting C2a and/or the C2a portion of C2 for complete inhibition ofthe classical and the lectin complement pathways has several advantagesincluding, for example: (1) C2 and C2a are specific for the classicaland the lectin complement pathways, and thus inhibition of C2 and/or C2awould achieve complete and selective inhibition of these two complementpathways without affecting the alternative complement pathway; (2) theconcentration of C2 in human blood is one of the lowest (ca. 20 μg/ml)among other soluble complement components, therefore inhibitors of C2 orC2a would have a unique dose advantage; and (3) since C2a is thecatalytic subunit of the C3 and C5 convertases, inhibition of C2 or theC2a portion of C2 would block the activation of C3 and C5.

The down-regulation of complement activation has been demonstrated to beeffective in treating several disease indications in animal models andin ex vivo studies, e.g., systemic lupus erythematosus andglomerulonephrtis (Y. Wang et al., Proc. Natl. Acad. Sci. (USA), 1996,93: 8563-8568), rheumatoid arthritis (Y. Wang et al., Proc. Natl. Acad.Sci.(USA), 1995, 92: 8955-8959), in preventing inflammation associatedwith cardiopulmonary bypass and hemodialysis (C. S. Rinder et al., J.Clin. Invest., 1995, 96:1564-1572; J. C. K. Fitch et al., Circulation,1999, 100: 2499-2506; H. L. Lazar et al., Circulation, 1999, 100:1438-1442), hyperacute rejection in organ transplantation (T. J. Kroshuset al., Transplantation, 1995, 60: 1194-1202), myocardial infarction (J.W. Homeister et al., J. Immunol., 1993, 150: 1055-1064; H. F. Weisman etal., Science, 1990, 249: 146-151), reperfusion injury (E. A. Amsterdamet al., Am. J. Physiol., 1995, 268: H448-H457), and adult respiratorydistress syndrome (R. Rabinovici et al., J. Immunol.,1992, 149:1744-1750). In addition, other inflammatory conditions andautoimmune/immune complex diseases are also closely associated withcomplement activation (V. M. Holers, ibid., B. P. Morgan. Eur. J. Clin.Invest, 1994, 24: 219-228), including thermal injury, severe asthma,anaphylactic shock, bowel inflammation, urticaria, angioedema,vasculitis, multiple sclerosis, psoriasis, dermatomyositis, myastheniagravis, membranoproliferative glomerulonephritis, and Sjögren'ssyndrome.

SUMMARY OF THE INVENTION

The present invention includes inhibitor molecules having a bindingregion specific for C2a or the C2a portion of C2. The inhibitor moleculemay be an antibody or a homologue, analogue or fragment thereof, apeptide, an oligonucleotide, a peptidomimetic or an organic compound.Antibody fragments can be Fab, F(ab′)₂, Fv or single chain Fv. Theinhibitor molecule may be in the form of a pharmaceutical composition.

One embodiment of the present invention includes an inhibitor moleculecomprising a monoclonal antibody. The antibody may be chimeric,DEIMMUNIZED™ (described below), humanized or human antibody.Specifically, the monoclonal antibody may be the monoclonal antibodydesignated 175-62.

Another embodiment of the invention is a hybridoma producing themonoclonal antibody 175-62.

Another embodiment of the invention includes monoclonal antibodies or afragment, analogue or homologue thereof, or a peptide, oligonucleotide,peptidomimetic or an organic compound which bind to the same epitope asthe antibody 175-62. These antibodies can include Fab, F(ab′)₂ Fv orsingle chain Fv, and may be chimeric, DEIMMUNIZED™, humanized or humanantibody. In addition, the present invention includes cell lines thatproduces the monoclonal antibody or fragment thereof that bind to thesame epitope the antibody 175-62.

The present invention also includes molecules that inhibit complementactivation by inhibiting both the classical and lectin complementpathways. The preferred molecules of the present invention inhibitcomplement activation at a molar ratio of inhibitor molecule to C2 at1:2.

Another embodiment of the present invention includes a method oftreating a disease or condition that is mediated by excessive oruncontrolled activation of the complement system by administering, invivo or ex vivo, an inhibitor molecule that specifically binds C2a orthe C2a portion of C2.

One example of a Mab, designated 175-62, that binds to C2a and blocksits ability to activate complement was generated as described below. Thehybridoma producing this antibody was deposited at the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,under Accession Number PTA-1553, on Mar. 22, 2000.

FIG. 1 shows the binding of anti-C2a MAbs (175 series), anti-C5 Mab(137-76), and anti-factor D Mab (166-32) to purified human C2a in anELISA. The Y-axis represents the reactivity of the MAbs with C2aexpressed as optical density (OD) at 450 nm and the X-axis representsthe concentration of the MAbs. MAb 175-62 shows the strongest reactivitywith C2a.

FIG. 2 shows the inhibition of classical pathway hemolysis of sensitizedchicken red blood cells (RBCs) by anti-C2a MAbs in the presence of 3%human serum. The controls were anti-factor D Mab (166-32) and theanti-C5 MAb (137-76). Anti-factor D Mab 166-32 specifically inhibits thealternative complement pathway, therefore it does not inhibit theclassical pathway hemolysis. The Y-axis represents the % hemolysisinhibition, as further described in the text. The X-axis represents theconcentration of the MAbs. All anti-C2a MAbs strongly inhibit classicalpathway hemolysis.

FIG. 3 shows that anti-C2a MAb 175-62 inhibits classical pathway (CP)hemolysis at a molar ratio of 1:2 (MAb 175-62 to C2). The filled circlesrepresent MAb 175-62. The open squares represent hemolysis in theabsence of MAb 175-62. The Y-axis represents the % hemolysis inhibition.The X-axis represents the concentration of serum. The classical pathwayhemolytic activity of C2 (0.2 μM) in normal human serum is completelyinhibited when the serum was pre-treated with 0.1 μM of MAb 175-62.

FIG. 4 shows an assay for testing the inhibition of alternative pathway(AP) hemolysis of unsensitized rabbit RBCs by anti-C2a, anti-factor Dand anti-C5 MAbs, in the presence of 10% human serum. The Y-axisrepresents the % hemolysis inhibition, as further described in the text.The X-axis represents the concentration of the MAbs. The data illustratethat none of the anti-C2a MAbs inhibit the alternative complementpathway.

DETAILED DESCRIPTION

The inhibitor molecules of the present invention include monoclonalantibodies as well as homologues, analogues and modified or derivedforms thereof, including immunoglobulin fragments such as Fab, F(ab′)₂,and Fv, and single chain antibodies. Also included are small moleculesincluding peptides, oligonucleotides, peptidomimetics and organiccompounds.

One embodiment of the invention includes anti-C2a MAbs, which can beraised by immunizing rodents (e.g. mice, rats, hamsters and guinea pigs)with either (1) native C2a derived from enzymatic digestion of C2purified from human plasma or serum, or (2) recombinant C2a or itsfragments expressed by either eukaryotic or prokaryotic systems. Otheranimals can be used for immunization, e.g. non-human primates,transgenic mice expressing human immunoglobulins, and severe combinedimmunodeficient (SCID) mice transplanted with human B-lymphocytes.

Hybridomas can be generated by conventional procedures by fusingB-lymphocytes from the immunized animals with myeloma cells (e.g., Sp2/0and NS0), as described by G. Köhler and C. Milstein (Nature, 1975, 256:495-497). In addition, anti-C2a antibodies can be generated by screeningrecombinant single-chain Fv or Fab libraries from human B-lymphocytes ina phage-display system. The specificity of the MAbs to human C2a can betested by enzyme linked immunosorbent assay (ELISA), Westernimmunoblotting, or other immunochemical techniques.

The inhibitory activity on complement activation of antibodiesidentified in the screening process can be assessed by hemolytic assaysusing either unsensitized rabbit or guinea pig RBCs for the alternativecomplement pathway, or sensitized chicken or sheep RBCs for theclassical complement pathway. Those hybridomas that exhibit aninhibitory activity specific for the classical complement pathway arecloned by limiting dilution. The antibodies are purified forcharacterization for specificity to human C2a by the assays describedabove.

When treating inflammatory or autoimmune diseases in humans, theanti-C2a antibodies may be chimeric, DEIMMUNIZED™, humanized or humanantibodies. Such antibodies can reduce immunogenicity, thereby avoidinga human/anti-mouse antibody (HAMA) response. It is preferable that theantibody be IgG4, IgG2, or other genetically mutated IgG or IgM whichdoes not augment antibody-dependent cellular cytotoxicity (S. M.Canfleid et al., J. Exp. Med., 1991, 173: 1483-1491) and complementmediated cytolysis (Y. Xu et al., J. Biol. Chem., 1994, 289: 3488-3474;V. L. Pulito et al., J. Immunol. 1996, 158:2840-2850).

Chimeric antibodies are produced by recombinant processes well known inthe art, and have an animal variable region and a human constant region.Humanized antibodies have a greater degree of human peptide sequencesthan do chimeric antibodies. In a humanized antibody, only thecomplementarity determining regions (CDRs), which are responsible forantigen binding and specificity, are animal derived. The amino acidsequence corresponding to the animal antibody, and substantially all ofthe remaining portions of the molecule (except, in some cases, smallportions of the framework regions within the variable region) are humanderived and correspond in amino acid sequence to a human antibody. See,e.g., L. Riechmann et al., Nature, 1988, 332: 323-327; G. Winter, U.S.Pat. No. 5,225,539; C. Queen et al., U.S. Pat. No. 5,530,101.

DEIMMUNIZED™ antibodies are antibodies in which the T-helper epitopeshave been eliminated, as described in international Patent ApplicationPCT/GB98/01473. They have either reduced or no immunogenicity whenadministered in vivo.

Human antibodies can be made by several different methods, including theuse of human immunoglobulin expression libraries (Stratagene Corp., LaJolla, Calif.) to produce fragments of human antibodies (VH, VL, Fv, Fd,Fab, or F(ab′)₂) to construct whole human antibodies using techniquessimilar to those for producing chimeric antibodies. Human antibodies canalso be produced in transgenic mice with a human immunoglobulin genome.Such mice are available from Abgenix, Inc., Fremont, Calif., andMedarex, Inc., Annandale, N.J.

One can also create single peptide chain binding molecules in which theheavy and light chain Fv regions are connected. Single chain antibodies(“scFv”) and the method of their construction are described in U.S. Pat.No. 4,946,778. Alternatively, Fab can be constructed and expressed bysimilar means (M. J. Evans et al., J. Immunol. Meth., 1995, 184:123-138).

Antibodies, fragments thereof, and single chain antibodies that arewholly or partially derived from human are less immunogenic than whollymurine MAbs, and therefore, less likely to evoke an immune or allergicresponse. Consequently, human-derived antibodies are better suited forin vivo administration in humans than wholly animal antibodies,especially when repeated or long-term administration is necessary. Inaddition, smaller size antibody fragments may help improve tissuebioavailability, which may offer better dose accumulation in certaindisease indications.

Based on the molecular structures of the variable regions of theanti-C2a antibodies, one can use molecular modeling and rationalmolecular design to generate and screen small molecules that mimic themolecular structures of the binding region of the antibodies and inhibitthe activities of C2a. These small molecules can be peptides,peptidomimetics, oligonucleotides, or organic compounds. The mimickingmolecules can be used as inhibitors of complement activation ininflammatory indications and autoimmune diseases. Alternatively, one canuse large-scale screening procedures commonly used in the field toisolate suitable small molecules from libraries of combinatorialcompounds.

Applications of the Anti-C2a Molecules

The anti-C2a binding molecules, antibodies, and fragments of the presentinvention can be administered to patients in an appropriatepharmaceutical formulation by a variety of routes, including, but notlimited, intravenous infusion, intravenous bolus injection, andintraperitoneal, intradermal, intramuscular, subcutaneous, intranasal,intratracheal, intraspinal, intracranial, and oral routes. Suchadministration enables them to bind to endogenous C2a or C2 and thusinhibit the generation of C3b, C3a and C5a anaphylatoxins, and C5b-9.

The estimated dosage of such antibodies and molecules is between 10 and500 μg/ml of serum. The actual dosage can be determined in clinicaltrials following the conventional methodology for determining optimaldosages, i.e., administering various dosages and determining which ismost effective.

The anti-C2a inhibitor molecules can function to inhibit in vivocomplement activation and inflammatory manifestations that accompany it,such as recruitment and activation of macrophages, neutrophils,platelets, mast cells and endothelial cells, edema, and tissue damage.These inhibitor molecules can be used for treatment of diseases orconditions that are mediated by excessive or uncontrolled activation ofthe complement system. These include, but are not limited to: (1) tissuedamage due to ischemia-reperfusion following acute myocardialinfarction, aneurysm, stroke, hemorrhagic shock, crush injury, multipleorgan failure, hypovolemic shock and intestinal ischemia; (2)inflammatory disorders, such as, burns, endotoxemia and septic shock,adult respiratory distress syndrome, cardiopulmonary bypass,hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn'sdisease, psoriasis, dermomyositis, sickle cell anemia, poststreptococcalglomerulonephritis, and pancreatitis; (3) transplant rejections, suchas, hyperacute xenograft rejection; and (4) adverse drug reactions, suchas, drug allergy, IL-2 induced vascular leakage syndrome, andradiographic contrast media allergy. Autoimmune disorders including, butnot limited to, systemic lupus erythematosus, myasthenia gravis,rheumatoid arthritis, Alzheimer's disease and multiple sclerosis, mayalso be treated with the inhibitor molecules of the invention.

The anti-C2a inhibitor molecules can also be used diagnostically toascertain the presence of, or to measure, C2a in a tissue specimen or abody fluid sample, such as serum, plasma, urine or spinal fluid. In thisapplication, common assay formats can be used, such asimmunohistochemistry or ELISA, respectively. Such diagnostic tests couldbe useful in determining whether certain individuals are eitherdeficient in or overproduce C2a.

Animal Models of the Therapeutic Efficacy of C2a Inhibitors

The therapeutic activity of C2a inhibitor molecules in various diseaseindications described above can be confirmed by using available animalmodels for various inflammatory and autoimmune manifestations.

Animal models relevant to various complement-related clinical diseasesin humans can be used to confirm the in vivo efficacy of C2a inhibitors.These include, but are not limited to: myocardial ischemia/reperfusioninjury (H. F. Weisman et al., Science, 1990, 249: 146-151); myocardialinfarction (J. W. Homeister et al., J. Immunol., 1993, 150: 1055-1064),systemic lupus erythematosus and glomerulonephritis (S. K. Datta. Meth.Enzymol., 1988, 162:385-442; D. J. Salvant et al., Meth. Enzymol., 1988,162: 421-461), rheumatoid arthritis (Y. Wang et al., Proc. Natl. Acad.Sci. (USA), 1995, 92: 8955-8959), adult respiratory distress syndrome(R. Rabinovici et al., J. Immunol., 1992, 149: 1744-1750), hyperacuterejection in organ transplantation (T. J. Kroshus et al.,Transplantation, 1995, 60: 1194-1202), burn injury (M. S. Mulligan etal., J. Immunol., 1992, 148: 1479-1485), cardiopulmonary bypass (C. S.Rinder et al., J. Clin. Invest, 1995, 96: 1564-1572).

EXAMPLE 1 Generation of Anti-C2a Mab Hybridomas

Eight to twelve-week old male A/J mice (Harlan, Houston, Tex.) weresubcutaneously injected with 20 μg of C2a in complete Freund's adjuvant(Difco Laboratories, Detroit, Mich.) in 200 μl of phosphate-bufferedsaline (PBS) pH 7.4. The C2a was generated by enzymatic digestion usingC1s (Advanced Research Technologies, San Diego, Calif.) conjugated toCNBr-activated Sepharose 6MB (Pharmacia Biotech, Piscataway, N.J.),similar to the procedure described in T. J. Oglesby, J. Immunol, 1988,141: 926-931. The resulting C2a was then purified by passage through aSephadex-200 size-exclusion HPLC column. The C2a preparation was testedto be >95% pure by sodium dodecylsulphate (SDS)-polyacrylamide gelelectrophoresis (PAGE). C2 was purified from human serum (AdvancedResearch Technologies).

At two-week intervals, mice were twice injected subcutaneously with 20μg of C2a in incomplete Freund's adjuvant. Then, two weeks later, threedays prior to sacrifice, the mice were again injected intraperitoneallywith 20 μg of the same antigen in PBS.

For each hybridoma, single cell suspensions were prepared from thespleen of an immunized mouse and fused with Sp2/0 myeloma cells. 5×10⁸of the Sp2/0 and 5×10⁸ spleen cells were fused in a medium containing50% polyethylene glycol (M. W. 1450) (Kodak, Rochester, N.Y.) and 5%dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.). The cells werethen adjusted to a concentration of 1.5×10⁵ spleen cells per 200 μl ofthe suspension in Iscove medium (Gibco, Grand Island, N.Y.),supplemented with 10% fetal bovine serum, 100 units/ml of penicillin,100 μg/ml of streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and16 μM thymidine. Two hundred μl of the cell suspension were added toeach well of about fifty 96-well microculture plates. After about tendays, culture supernatants were withdrawn for screening for reactivitywith purified C2a in ELISA.

Wells of Immulon 2 (Dynatech Laboratories, Chantilly, Va.) microtestplates were coated by adding 50 μl of purified human C2a at 50 ng/mlovernight at room temperature. The low concentration of C2a used forcoating enabled the selection of high-affinity antibodies. After thecoating solution was removed by flicking the plate, 200 μl of BLOTTO(non-fat dry milk) in PBS was added to each well for one hour to blockthe non-specific sites. An hour later, the wells were then washed with abuffer PBST (PBS containing 0.05% Tween 20). Fifty microliters ofculture supernatants from each fusion well were collected and mixed with50 μl of BLOTTO and then added to the individual wells of the microtestplates. After one hour of incubation, the wells were washed with PBST.The bound murine antibodies were then detected by reaction withhorseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Fcspecific) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) anddiluted at 1:2,000 in BLOTTO. Peroxidase substrate solution containing0.1% 3,3,5,5 tetramethyl benzidine (Sigma) and 0.0003% hydrogen peroxide(Sigma) was added to the wells for color development for 30 minutes. Thereaction was terminated by addition of 50 μl of 2M H₂SO₄ per well. TheOD at 450 nm of the reaction mixture was read with a BioTek ELISA Reader(BioTek Instruments, Winooski, Vt.).

The culture supernatants from the positive wells were then tested forinhibition of classical pathway hemolysis of sensitized chicken RBCs bypre-titered human serum (3%) by the method described below. The cells inthose positive wells were cloned by limiting dilution. The MAbs weretested again for reactivity with C2a and C2 in the ELISA. The selectedhybridomas were grown in spinner flasks and the spent culturesupernatant collected for antibody purification by protein A affinitychromatography. Ten MAbs were tested to be reactive with human C2a inELISA. These MAbs are designated MAbs 175-50, 175-62, 175-97-1,175-97-4, 175-101, 175-207, 175-283, 175-310, 175-322, and 175-326. Asseen in FIG. 1, MAb 175-62, MAb 175-101, MAb 175-207, MAb 175-310, MAb175-322, and MAb 175-326 reacted strongly with human C2a in ELISA. Inparticular, MAb 175-62 shows the strongest reactivity with C2a amongthese binders. Interestingly, it binds weakly to immobilized C2 inELISA.

EXAMPLE 2 Inhibition of Complement-activated Hemolysis

To study the functional activity of the anti-C2a MAbs in inhibitingcomplement activation in vitro, two hemolytic assays were used.

For the classical pathway, chicken RBCs (5×10⁷ cells/ml), ingelatin/veronal-buffered saline (GVB⁺⁺) containing 0.5 mM MgCl₂ and 0.15mM CaCl₂, were sensitized with purified rabbit anti-chicken RBCimmunoglobulins at 8 μg/ml (Inter-Cell Technologies, Hopewell, N.J.) for15 minutes at 4° C. The cells were then washed with GVB⁺⁺. The washedcells were re-suspended in the same buffer at 1.7×10⁸ cells/ml. In eachwell of a round-bottom 96-well microtest plate, 50 μl of normal humanserum (6%) was mixed with 50 μl of GVB⁺⁺ or serially diluted test MAb,then 30 μl of the washed sensitized chicken RBC suspension were added tothe wells containing the mixtures. Fifty microliters of normal humanserum (6%) was mixed with 80 μl of GVB⁺⁺ to give the serum colorbackground. The final mixture was incubated at 37° C. for 30 minutes.The plate was then shaken on a micro-test plate shaker for 15 seconds,followed by centrifugation at 300×g for 3 minutes. Supernatants (80 μl)were collected and transferred to wells on a flat-bottom 96-wellmicrotest plates for measurement of OD at 405 nm. The percent inhibitionof hemolysis is defined as 100×[(OD without MAb−OD serum colorbackground)−(OD with MAb−OD serum color background)]/(OD without MAb−ODserum color background).

The data in FIG. 2 show that the anti-C2a MAbs 175-62, 175-207, 175-310,175-322, and 175-326 strongly inhibit classical pathway hemolysis. Theanti-C5 MAb 137-76 also inhibits the hemolysis, but not the anti-factorD MAb 166-32, which is specific for inhibition of the alternativecomplement pathway.

The stoichiometric ratio of inhibition between MAb 175-62 and C2 inhuman serum by the classical pathway hemolytic assays was also measuredas described above. Different molar ratios of MAb 175-62 to C2 weretested in the assays by combining normal human serum (containing 20μg/ml or 0.2 μM of C2) with 0.4 μM, 0.2 μM, or 0.1 μM of MAb 175-62. Thecontrol was normal human serum treated with equal volume of GVB⁺⁺. Themixtures were incubated at room temperature for 15 minutes. The mixtureswere then serially diluted in GVB⁺⁺. One hundred microliters of thediluted serum samples were added to each well of a round-bottom 96-wellplate in duplicate. Thirty microliters of sensitized chicken RBCs werethen added to each well for incubation as described above. The finalmixture was incubated at 37° C. for 30 minutes. The plate was thenshaken on a micro-test plate shaker for 15 seconds, followed bycentrifugation at 300×g for 3 minutes. Supernatants (80 μl) werecollected and transferred to wells on a flat-bottom 96-well microtestplates for measurement of OD at 405 nm.

The data in FIG. 3 show that the classical pathway hemolytic activity ofC2 (0.2 μM) in normal human serum is completely inhibited when the serumwas pre-treated with 0.1 μM of MAb 175-62. Therefore, MAb 175-62inhibits human C2 at a molar ratio of 1:2 (MAb 175-62 to C2). In otherwords, MAb 175-62 is a very high-affinity anti-C2 antibody. Each of thetwo antigen binding sites in a molecule of MAb 175-62 can bind onemolecule of C2.

For the alternative pathway, unsensitized rabbit RBCs were washed threetimes with gelatin/veronal-buffered saline (GVB/Mg-EGTA) containing 2 mMMgCl₂ and 1.6 mM EGTA. EGTA at a concentration of 10 mM was used toinhibit the classical pathway (K. Whaley et al., in A. W. Dodds (Ed.),Complement: A Practical Approach. Oxford University Press, Oxford, 1997,pp. 19-47). The procedures of the assay were similar to those of theclassical pathway hemolysis as described above. The final concentrationof human serum was 10%.

The data in FIG. 4 show that none of the anti-C2a MAbs inhibit thealternative pathway hemolysis, whereas anti-factor D MAb 166-32effectively inhibits the hemolysis and anti-C5 MAb 137-76 moderatelyinhibits the hemolysis. Together with the results in FIGS. 2 and 3, theanti-C2a MAbs have been shown to be specific for the classicalcomplement pathway.

1. An isolated antibody that binds to the C2a fragment of complementprotein C2 or the C2a portion of C2, or a C2a binding fragment thereof,which inhibits complement activation more than 50% at a molar radio ofantibody to C2 of 1:2.
 2. The antibody or C2a, binding fragment thereofof claim 1, wherein the antibody fragment is a Fab, F(ab′)₂, Fv orsingle chain Fv.
 3. The antibody of C2a binding fragment thereof ofclaim 1, wherein the antibody is monoclonal.
 4. The antibody or C2abinding fragment thereof of claim 3, wherein the antibody is a chimeric,deimmunized, humanized or a human antibody.
 5. A composition comprisingthe antibody or C2a binding fragment thereof of claim 1 and apharmacologically acceptable carrier, excipient, stabilizer, or diluent.6. A diagnostic method comprising the detection of the amount of C2 orC2a present in a sample with the antibody or C2a binding fragmentthereof of claim
 1. 7. The diagnostic method of claim 6, wherein theantibody is the monoclonal antibody designated 175-62 and produced bythe hybridoma deposited under ATCC Accession number PTA-1553.
 8. Anisolated antibody that binds to the C2a fragment of component protein C2or the C2a portion of C2, or a C2a binding fragment thereof, whichinhibits both the classical and the lectin complement pathways more than50% at a molar ratio of antibody to C2 of 1:2.
 9. A monoclonal antibodydesignated 175-62 produced by the hybridoma cell line deposited underATCC Accession Number PTA-1553.
 10. A cell line that produces themonoclonal antibody designated 175-62, said cell line deposited underATCC Accession No. PTA-1553.
 11. An isolated antibody that binds to theC2a fragment of complement protein C2 or the C2a portion of C2, or a C2abinding fragment thereof, which completely inhibits complementactivation at a molar ratio of antibody to C2 of 1:2.
 12. The antibodyor C2a binding fragment thereof of claim 11, wherein the isolatedantibody is a monoclonal antibody designated 175-62 produced by thehybridoma cell line deposited under ATCC Accession Number PTA-1553.